Huh7.5 cells were plated in a 24-well plate, 18 to 24 h prior to transfection. Fresh culture medium was added to each well 1 h before transfection. Thereafter, the cells were transfected with 0.5 μg pcDNA3.1+/C-(K)DYK-ACE2 (GenScript) diluted in 25 μL serum-free DMEM, or mock transfected, and PolyJet reagent (SignaGen Laboratories), according to the manufacturer’s instructions. Medium was replaced after 6 h and 48 h posttransfection, both transfected and non-transfected cells were infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.
For knockdown experiments, Huh7.5 cells were seeded in a 24-well plate and incubated until they reached 80% confluence, which was generally 24 h after seeding. Cells were then transfected with ON-TARGETplus Human ACE2 siRNA (Dharmacon) or Non-targeting Pool (Dharmacon) using RNAiMAX (Invitrogen), incubated for 24 h and then infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.
For knockdown experiments, Huh7.5 cells were seeded in a 24-well plate and incubated until they reached 80% confluence, which was generally 24 h after seeding. Cells were then transfected with ON-TARGETplus Human ACE2 siRNA (Dharmacon) or Non-targeting Pool (Dharmacon) using RNAiMAX (Invitrogen), incubated for 24 h and then infected with HCV at an MOI of 0.1. Cells were harvested 72 h postinfection for RNA isolation.